Nucleotide regulation of the voltage-dependent nonselective cation conductance in murine colonic myocytes

2006 ◽  
Vol 291 (5) ◽  
pp. C985-C994 ◽  
Author(s):  
Kevin P. Monaghan ◽  
Sang Don Koh ◽  
Seungil Ro ◽  
Jonghun Yeom ◽  
Burton Horowitz ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Phospholipase C ◽  
Threshold Voltage ◽  
Pyridoxal Phosphate ◽  
Disulfonic Acid ◽  
Smooth Muscle Relaxation ◽  
External Application ◽  
Voltage Dependent ◽  
Inward Currents ◽  
Stimulation Of

ATP is proposed to be a major inhibitory neurotransmitter in the gastrointestinal (GI) tract, causing hyperpolarization and smooth muscle relaxation. ATP activates small-conductance Ca2+-activated K+ channels that are involved in setting the resting membrane potential and causing inhibitory junction potentials. No reports are available examining the effects of ATP on voltage-dependent inward currents in GI smooth muscle cells. We previously reported two types of voltage-dependent inward currents in murine proximal colonic myocytes: a low-threshold voltage-activated, nonselective cation current ( IVNSCC) and a relatively high-threshold voltage-activated (L-type) Ca2+ current ( IL). Here we have investigated the effects of ATP on these currents. External application of ATP (1 mM) did not affect IVNSCC or IL in dialyzed cells. ATP (1 mM) increased IVNSCC and decreased IL in the perforated whole-cell configuration. UTP and UDP (1 mM) were more potent than ATP on IVNSCC. ADP decreased IL but had no effect on IVNSCC. The order of effectiveness was UTP = UDP > ATP > ADP. These effects were not blocked by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS), but the phospholipase C inhibitor U-73122 reversed the effects of ATP on IVNSCC. ATP stimulation of IVNSCC was also reversed by protein kinase C (PKC) inhibitors chelerythrine chloride or bisindolylmaleimide I. Phorbol 12,13-dibutyrate mimicked the effects of ATP. RT-PCR showed that P2Y4 is expressed by murine colonic myocytes, and this receptor is relatively insensitive to PPADS. Our data suggest that ATP activates IVNSCC and depresses IL via binding of P2Y4 receptors and stimulation of the phospholipase C/PKC pathway.

Membrane currents in canine bronchial artery and their regulation by excitatory agonists

2002 ◽  
Vol 282 (6) ◽  
pp. L1358-L1365 ◽  
Author(s):  
Q. J. Li ◽  
L. J. Janssen
Keyword(s):  
Smooth Muscle ◽  
Bronchial Artery ◽  
Similar Degree ◽  
Ion Currents ◽  
Significant Suppression ◽  
Bronchial Smooth Muscle ◽  
Voltage Dependent ◽  
Inward Currents ◽  
Bronchial Smooth Muscle Cells ◽  
Airway Physiology

The bronchial vasculature plays an important role in airway physiology and pathophysiology. We investigated the ion currents in canine bronchial smooth muscle cells using patch-clamp techniques. Sustained outward K+current evoked by step depolarizations was significantly inhibited by tetraethylamonium (1 and 10 mM) or by charybdotoxin (10−6M) but was not significantly affected by 4-aminopyridine (1 or 5 mM), suggesting that it was primarily a Ca2+-activated K+current. Consistent with this, the K+current was markedly increased by raising external Ca2+to 4 mM but was decreased by nifedipine (10−6M) or by removing external Ca2+. When K+currents were blocked (by Cs+in the pipette), step depolarizations evoked transient inward currents with characteristics of L-type Ca2+current as follows: 1) activation that was voltage dependent (threshold and maximal at −50 and −10 mV, respectively); 2) inactivation that was time dependent and voltage dependent (voltage causing 50% maximal inactivation of −26 ± 22 mV); and 3) blockade by nifedipine (10−6M). The thromboxane mimetic U-46619 (10−6M) caused a marked augmentation of outward K+current (as did 10 mM caffeine) lasting only 10–20 s; this was followed by significant suppression of the K+current lasting several minutes. Phenylephrine (10−4M) also suppressed the K+current to a similar degree but did not cause the initial transient augmentation. None of these three agonists elicited inward current of any kind. We conclude that bronchial arterial smooth muscle expresses Ca2+-dependent K+channels and voltage-dependent Ca2+channels and that its excitation does not involve activation of Cl−channels.

scholarly journals Small-conductance Ca2+-dependent K+ channels activated by ATP in murine colonic smooth muscle

1997 ◽  
Vol 273 (6) ◽  
pp. C2010-C2021 ◽  
Author(s):  
S. D. Koh ◽  
G. M. Dick ◽  
K. M. Sanders
Keyword(s):  
Smooth Muscle ◽  
Pyridoxal Phosphate ◽  
Single Channel ◽  
Outward Current ◽  
Disulfonic Acid ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Outward Currents ◽  
Murine Colon ◽  
Small Conductance

The patch-clamp technique was used to determine the ionic conductances activated by ATP in murine colonic smooth muscle cells. Extracellular ATP, UTP, and 2-methylthioadenosine 5′-triphosphate (2-MeS-ATP) increased outward currents in cells with amphotericin B-perforated patches. ATP (0.5–1 mM) did not affect whole cell currents of cells dialyzed with solutions containing ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid. Apamin (3 × 10−7M) reduced the outward current activated by ATP by 32 ± 5%. Single channel recordings from cell-attached patches showed that ATP, UTP, and 2-MeS-ATP increased the open probability of small-conductance, Ca2+-dependent K+ channels with a slope conductance of 5.3 ± 0.02 pS. Caffeine (500 μM) enhanced the open probability of the small-conductance K+ channels, and ATP had no effect after caffeine. Pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid tetrasodium (PPADS, 10−4 M), a nonselective P2 receptor antagonist, prevented the increase in open probability caused by ATP and 2-MeS-ATP. PPADS had no effect on the response to caffeine. ATP-induced hyperpolarization in the murine colon may be mediated by P2y-induced release of Ca2+ from intracellular stores and activation of the 5.3-pS Ca2+-activated K+ channels.

Effect of exogenous ATP on canine jejunal smooth muscle

2000 ◽  
Vol 278 (5) ◽  
pp. G725-G733 ◽  
Author(s):  
L. Xue ◽  
G. Farrugia ◽  
J. H. Szurszewski
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Pyridoxal Phosphate ◽  
Electrical Field Stimulation ◽  
Muscle Cells ◽  
Disulfonic Acid ◽  
Reactive Blue 2 ◽  
Circular Smooth Muscle ◽  
Inhibitory Junction Potential ◽  
Junction Potential

Intracellular recordings were made from the circular smooth muscle cells of the canine jejunum to study the effect of exogenous ATP and to compare the ATP response to the nonadrenergic, noncholinergic (NANC) inhibitory junction potential (IJP) evoked by electrical field stimulation (EFS). Under NANC conditions, exogenous ATP evoked a transient hyperpolarization (6.5 ± 0.6 mV) and EFS evoked a NANC IJP (17 ± 0.4 mV). ω-Conotoxin GVIA (100 nM) and a low-Ca2+, high-Mg2+ solution abolished the NANC IJP but had no effect on the ATP-evoked hyperpolarization. The ATP-evoked hyperpolarization and the NANC IJP were abolished by apamin (1 μM) and N G-nitro-l-arginine (100 μM). Oxyhemoglobin (5 μM) partially (38.8 ± 5.5%) reduced the amplitude of the NANC IJP but had no effect on the ATP-evoked hyperpolarization. Neither the NANC IJP nor the ATP-evoked hyperpolarization was affected by P2 receptor antagonists or agonists, including suramin, reactive blue 2, 1-( N, O-bis-[5-isoquinolinesulfonyl]- N-methyl-l-tyrosyl)-4-phenylpiperazine, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, α,β-methylene ATP, 2-methylthioadenosine 5′-triphosphate tetrasodium salt, and adenosine 5′- O-2-thiodiphosphate. The data suggest that ATP evoked an apamin-sensitive hyperpolarization in circular smooth muscle cells of the canine jejunum via local production of NO in a postsynaptic target cell.

scholarly journals Functional characterization of voltage-dependent Ca2+ channels in mouse pulmonary arterial smooth muscle cells: divergent effect of ROS

2013 ◽  
Vol 304 (11) ◽  
pp. C1042-C1052 ◽  
Author(s):  
Eun A. Ko ◽  
Jun Wan ◽  
Aya Yamamura ◽  
Adriana M. Zimnicka ◽  
Hisao Yamamura ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Function ◽  
Electromechanical Coupling ◽  
Functional Characterization ◽  
Muscle Cells ◽  
Pulmonary Vascular Disease ◽  
High K ◽  
Voltage Dependent ◽  
Inward Currents

Electromechanical coupling via membrane depolarization-mediated activation of voltage-dependent Ca2+ channels (VDCC) is an important mechanism in regulating pulmonary vascular tone, while mouse is an animal model often used to study pathogenic mechanisms of pulmonary vascular disease. The function of VDCC in mouse pulmonary artery (PA) smooth muscle cells (PASMC), however, has not been characterized, and their functional role in reactive oxygen species (ROS)-mediated regulation of vascular function remains unclear. In this study, we characterized the electrophysiological and pharmacological properties of VDCC in PASMC and the divergent effects of ROS produced by xanthine oxidase (XO) and hypoxanthine (HX) on VDCC in PA and mesenteric artery (MA). Our data show that removal of extracellular Ca2+ or application of nifedipine, a dihydropyridine VDCC blocker, both significantly inhibited 80 mM K+-mediated PA contraction. In freshly dissociated PASMC, the maximum inward Ca2+ currents were −2.6 ± 0.2 pA/pF at +10 mV (with a holding potential of −70 mV). Window currents were between −40 and +10 mV with a peak at −15.4 mV. Nifedipine inhibited currents with an IC50 of 0.023 μM, and 1 μM Bay K8644, a dihydropyridine VDCC agonist, increased the inward currents by 61%. XO/HX attenuated 60 mM K+-mediated increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) due to Ca2+ influx through VDCC in PASMC. Exposure to XO/HX caused relaxation in PA preconstricted by 80 mM K+ but not in aorta and MA. In contrast, H2O2 inhibited high K+-mediated increase in [Ca2+]cyt and caused relaxation in both PA and MA. Indeed, RT-PCR and Western blot analysis revealed significantly lower expression of CaV1.3 in MA compared with PA. Thus our study characterized the properties of VDCC and demonstrates that ROS differentially regulate vascular contraction by regulating VDCC in PA and systemic arteries.

scholarly journals Exocytotic release of ATP and activation of P2X receptors in dissociated guinea pig stellate neurons

2006 ◽  
Vol 291 (5) ◽  
pp. C1062-C1071 ◽  
Author(s):  
John D. Tompkins ◽  
Rodney L. Parsons
Keyword(s):  
Guinea Pig ◽  
Pyridoxal Phosphate ◽  
Purinergic Receptor ◽  
P2x Receptors ◽  
Disulfonic Acid ◽  
Inward Current ◽  
Protein Receptor ◽  
Vesicular Release ◽  
Cell Recording ◽  
Inward Currents

Activation of P2X receptors by a Ca2+- and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent release of ATP was measured using patch-clamp recordings from dissociated guinea pig stellate neurons. Asynchronous transient inward currents (ASTICs) were activated by depolarization or treatment with the Ca2+ ionophore ionomycin (1.5 and 3 μM). During superfusion with a HEPES-buffered salt solution containing 2.5 mM Ca2+, depolarizing voltage steps (−60 to 0 mV, 500 ms) evoked ASTICs on the decaying phase of a larger, transient inward current. Equimolar substitution of Ba2+ for Ca2+ augmented the postdepolarization frequency of ASTICs, while eliminating the larger transient current. Perfusion with an ionomycin-containing solution elicited a sustained activation of ASTICs, allowing quantitative analysis over a range of holding potentials. Under these conditions, increasing extracellular [Ca2+] to 5 mM increased ASTIC frequency, whereas no events were observed following replacement of Ca2+ with Mg2+, demonstrating a Ca2+ requirement. ASTICs were Na+ dependent, inwardly rectifying, and reversed near 0 mV. Treatment with the nonselective purinergic receptor antagonist pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) (10 μM) blocked all events under both conditions, whereas the ganglionic nicotinic antagonist hexamethonium (100 μM and 1 mM) had no effect. PPADS also blocked the macroscopic inward current evoked by exogenously applied ATP (300 μM). The presence of botulinum neurotoxin E (BoNT/E) in the whole-cell recording electrode significantly attenuated the ionomycin-induced ASTIC activity, whereas phorbol ester treatment potentiated this activity. These results suggest that ASTICs are mediated by vesicular release of ATP and activation of P2X receptors.

Activation of thin-fiber muscle afferents by a P2X agonist in cats

2004 ◽  
Vol 96 (3) ◽  
pp. 1166-1169 ◽  
Author(s):  
Ramy L. Hanna ◽  
Marc P. Kaufman
Keyword(s):  
Pyridoxal Phosphate ◽  
Muscle Afferents ◽  
Group Iv ◽  
Triceps Surae ◽  
Disulfonic Acid ◽  
P2 Receptor ◽  
Thin Fiber ◽  
Group Iii ◽  
Decerebrate Cats ◽  
Stimulation Of

The responses of group III and IV triceps surae muscle afferents to intra-arterial injection of α,β-methylene ATP (50 μg/kg) was examined in decerebrate cats. We found that this P2X3 agonist stimulated only three of 18 group III afferents but 7 of 9 group IV afferents ( P < 0.004). The three group III afferents stimulated by α,β-methylene ATP conducted impulses below 4 m/s. Pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, a P2-receptor antagonist, prevented the stimulation of these afferents by α,β-methylene ATP. We conclude that P2X3 agonists stimulate only the slowest conducting group III muscle afferents as well as group IV afferents.

Chemical profile of vagal preganglionic motor cells innervating the airways in ferrets: the absence of noncholinergic neurons

2004 ◽  
Vol 97 (4) ◽  
pp. 1508-1517 ◽  
Author(s):  
Prabha Kc ◽  
Catherine A. Mayer ◽  
Musa A. Haxhiu
Keyword(s):  
Smooth Muscle ◽  
Muscarinic Receptor ◽  
Airway Smooth Muscle ◽  
Muscle Tone ◽  
Smooth Muscle Relaxation ◽  
Receptor Blockade ◽  
Double Labeling ◽  
Smooth Muscle Tone ◽  
Preganglionic Neurons ◽  
Stimulation Of

In ferrets, we investigated the presence of choline acetyltransferase (ChAT), vasoactive intestinal peptide (VIP), and markers for nitric oxide synthase (NOS) in preganglionic parasympathetic neurons innervating extrathoracic trachea and intrapulmonary airways. Cholera toxin β-subunit, a retrograde axonal transganglionic tracer, was used to identify airway-related vagal preganglionic neurons. Double-labeling immunohistochemistry and confocal microscopy were employed to characterize the chemical nature of identified airway-related vagal preganglionic neurons at a single cell level. Physiological experiments were performed to determine whether activation of the VIP and ChAT coexpressing vagal preganglionic neurons plays a role in relaxation of precontracted airway smooth muscle tone after muscarinic receptor blockade. The results showed that 1) all identified vagal preganglionic neurons innervating extrathoracic and intrapulmonary airways are acetylcholine-producing cells, 2) cholinergic neurons innervating the airways coexpress ChAT and VIP but do not contain NOS, and 3) chemical stimulation of the rostral nucleus ambiguus had no significant effect on precontracted airway smooth muscle tone after muscarinic receptor blockade. These studies indicate that vagal preganglionic neurons are cholinergic in nature and coexpress VIP but do not contain NOS; their stimulation increases cholinergic outflow, without activation of inhibitory nonadrenergic, noncholinergic ganglionic neurons, stimulation of which induces airway smooth muscle relaxation. Furthermore, these studies do not support the possibility of direct inhibitory innervation of airway smooth muscle by vagal preganglionic fibers that contain VIP.

Analysis of purine- and pyrimidine-induced vascular responses in the isolated rat cerebral arteriole

2001 ◽  
Vol 280 (2) ◽  
pp. H767-H776 ◽  
Author(s):  
Tetsuyoshi Horiuchi ◽  
Hans H. Dietrich ◽  
Shinichiro Tsugane ◽  
Ralph G. Dacey
Keyword(s):  
Smooth Muscle ◽  
Pyridoxal Phosphate ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Cerebral Microvessels ◽  
Vascular Responses ◽  
Single Receptor ◽  
Low Concentrations ◽  
High Concentrations ◽  
Cerebral Arteriole

Effects of extraluminal UTP were studied and compared with vascular responses to ATP and its analogs in rat cerebral-penetrating arterioles. UTP, UDP, 2-methylthio-ATP, and α,β-methylene-ATP dilated arterioles at the lowest concentration and constricted them at high concentrations. Low concentrations of ATP dilated the vessels; high concentrations caused a biphasic response, with transient constriction followed by dilation. Endothelial impairment inhibited ATP- and UTP-mediated dilation and potentiated constriction to UTP but not to ATP. ATP- and 2-methylthio-ATP- but not UTP-mediated constrictions were inhibited by desensitization with 10−6M α,β-methylene-ATP or 3 × 10−6M pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). PPADS at 10−4M abolished the UTP-mediated constriction and induced vasodilation in a dose-dependent manner but did not affect the dilation to ATP. These results suggest that in rat cerebral microvessels 1) ATP and 2-methylthio-ATP induce transient constriction via smooth muscle P2X1receptors in the cerebral arteriole, 2) UTP stimulates two different classes of P2Yreceptors, resulting in constriction (smooth muscle P2Y4) and dilation (possibly endothelial P2Y2), and 3) ATP and UTP produce dilation by stimulation of a single receptor (P2Y2).

Sign in / Sign up

Export Citation Format

Share Document